基于网络药理学探讨化浊行血汤抗高脂血症的作用机制

Exploration of the mechanism of Huazhuo Xingxue Decoction against hyperlipidemia based on network pharmacology

关键词:  网络药理学  化浊行血汤  高脂血症  作用机制

Key words:  network pharmacology  Huazhuo Xingxue Decoction  hyperlipidemia  mechanism

摘要:

[目的] 基于网络药理学探讨化浊行血汤抗高脂血症的作用机制。[方法] 通过高脂饲料喂养8周构建高脂血症大鼠模型。将大鼠分为对照组、模型组、化浊行血汤高剂量组、化浊行血汤中剂量组、化浊行血汤低剂量组、辛伐他汀组。灌胃8周后检测大鼠体质量、肝湿质量、肝指数以及血清胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）含量，苏木精-伊红（HE）染色观察大鼠肝脏组织学变化。借助中药系统药理学数据库与分析平台（TCMSP）及中药分子机制生物信息分析工具（BATMAN-TCM）筛选化浊行血汤的活性成分及相关靶点。借助药物银行（Drugbank）、疾病相关基因与突变位点数据库（DisGeNET）、比较毒理基因组数据库（CTD）、人类孟德尔遗传数据库（OMIM）及遗传关联数据库（GAD）查询高脂血症相关靶点。获取化浊行血汤及高脂血症交集靶点，在Cytoscape3.7.2中构建成分-靶点网络。将交集靶点导入String数据库获取蛋白互作（PPI）网络，对PPI网络进行拓扑分析获取关键靶点。将交集靶点导入基因注释形象集成数据库（DAVID）进行基因本体（GO）与京都基因与基因组百科全书（KEGG）富集分析。实时荧光定量聚合酶链式反应法（RT-PCR）测定关键靶点mRNA表达水平。[结果] 与对照组比较，模型组大鼠肝指数升高（P＜0.05），肝脂肪变性明显，血清TC，TG及LDL-C水平显著升高（P＜0.05）。与模型组比较，化浊行血汤各组大鼠肝指数降低（P＜0.05），肝脂肪变性明显改善，血清TC，TG及LDL-C含量降低（P＜0.05），化浊行血汤高中剂量组HDL-C含量升高（P＜0.05）。筛选获取化浊行血汤56个生物活性成分及327个靶点，高脂血症148个靶点。化浊行血汤与高脂血症交集靶点37个，涉及化浊行血汤37个生物活性成分，包括槲皮素、藏花酸、山柰酚、豆甾醇等。筛选获得胰岛素（INS）、三磷酸腺苷结合盒转运蛋白（ABCA1）、过氧化物酶体增殖物激活受体α（PPARA）、过氧化物酶体增殖物激活受体γ（PPARG）、脂联素（ADIPOQ）、载脂蛋白A2（APOA2）等15个关键靶点。富集分析预测化浊行血汤抗高脂血症可能涉及PPAR、腺苷酸活化蛋白激酶（AMPK）、磷脂酰肌醇3激酶-蛋白激酶B（PI3K-Akt）等信号通路。选取PPAR信号通路上的关键靶点脂联素受体2（AdipoR2）、PPARα及APOA2进行实验验证。与对照组比较，模型组AdipoR2和PPARα mRNA表达水平下调（P＜0.05）。与模型组比较，化浊行血汤高剂量组AdipoR2 mRNA表达水平上调（P＜0.05），化浊行血汤高中剂量组PPARα mRNA表达水平上调（P＜0.05），化浊行血汤高中剂量组APOA2 mRNA表达水平下调（P＜0.05）。[结论] 化浊行血汤能够改善高脂血症大鼠脂代谢紊乱，减轻脂质沉积，其抗高脂血症的机制可能与干预AdipoR2/PPARα/APOA2信号通路有关。

Abstract:

[Objective] To elucidate the mechanisms of Huazhuo Xingxue Decoction（HXD） against hyperlipidemia based on network pharmacology. [Methods] Rats model of hyperlipidemia was established by a high-fat diet for 8 weeks. The rats were divided into the control group，model group，high-dose group of HXD，medium-dose group of HXD，low-dose group of HXD and simvastatin group. After 8 weeks of intragastric administration，the weight，liver wet weight，liver index，serum TC，TG，LDL-C and HDL-C were measured. Pathomorphological changes in liver were observed by HE staining. The active ingredients and targets of HXD were retrieved from TCMSP and BATMAN-TCM. Related targets of hyperlipidemia were searched in Drugbank，DisGeNET，CTD，OMIM and GAD databases. Common targets of HXD and hyperlipidemia were extracted. Cytoscape 3.7.2 was used to set up the compound-target network. HXD-hyperlipidemia common target PPI network was established by using the STRING database and the topological parameters of PPI data were used to screen the hub targets. Then GO and KEGG enrichment analysis of common targets was carried out by David database. The mRNA expression were detected by RT-PCR. [Results] Compared with the control group，the liver index were higher（P＜0.05），liver steatosis were obvious，and serum TC，TG and LDL-C levels were significantly increased（P＜0.05）. Compared with the model group，the liver index were lower（P＜0.05），liver steatosis were attenuated significantly，and the serum TC，TG and LDL-C levels of rats in all groups of HXD were decreased（P＜0.05）；the HDL-C level increased in the high-dose group of HXD（P＜0.05）. Systems pharmacology revealed 327 targets for the 56 active ingredients of HXD and 112 candidate targets of hyperlipidemia. The 37 common targets of HXD and hyperlipidemia involved 37 active ingredients，such as crocetin，kaempferol，stigmasterol and physovenine. By screening，there were 15 hub targets were obtained，such as INS，ABCA1，PPARA，PPARG，ADIPOQ，APOA2. The enrichment analysis predicted that HXD had lipid-lowering effect mainly through PPAR，AMPK and PI3K/Akt signal pathway. AdipoR2，PPARα and APOA2 in PPAR signaling pathway were validated in PCR experiments. Compared with the control group，the mRNA expression of AdipoR2 and PPARα were down-regulated（P＜0.05） in the model group. Compared with the model group，the mRNA expression of AdipoR2 was up-regulated in the high-dose group of HXD（P＜0.05）；the mRNA expression of PPARα was up-regulated in the high-dose group and the medium-dose group of HXD（P＜0.05）；the mRNA expression of APOA2 was down-regulated in the high-dose group of HXD（P＜0.05）. [Conclusion] HXD can improve the disorder of lipid metabolism and reduce lipid deposition in hyperlipidemic rats，and its anti-hyperlipidemia mechanism may be related to the intervention of AdipoR2/ PPARα/APOA2 signaling pathway.